Acta crystallographica. Section F, Structural biology communications最新文献

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) regulates the level of cholesterol by catalysing the formation/production of mevalonate and has therefore become an important pharmaceutical target for coronary heart disease. Here, we report the cryo-EM structure of the catalytic part of the enzyme in the apo form and bound with its inhibitor atorvastatin, a commonly used drug in cardiovascular disease, at resolutions of 2.1 and 2.3 Å, respectively. In the cryo-EM maps, part of the N-domain corresponding to amino acids 439-487 is well ordered and could be modelled completely. Atorvastatin molecules were found to occupy all four active sites of the tetrameric complex, and the binding does not alter the conformation of the protein or the active site. The method described here exploits graphene oxide as an additional support and could be used as an alternative to elucidate the structures of pharmaceutical target compounds that are difficult to co-crystallize with human HMGR and for sparsely available samples in drug discovery.

High-resolution X-ray and neutron crystallography were employed to elucidate redox-dependent structural changes in ferredoxin-NADP⁺ reductase (FNR) from maize. This study focused on the rearrangement of hydrogen-bond networks upon FAD reduction. The X-ray structures of wild-type FNR in oxidized and reduced states were refined to 1.15 and 1.10 Å resolution, respectively, revealing no large structural changes in the main-chain backbones. Neutron crystallography provided complementary insights, confirming protonation at N1 and N5 of the isoalloxazine ring and visualizing hydrogen bonds that were undetectable by X-ray analysis. These findings illuminate the dynamic reorganization of water-mediated hydrogen-bond networks during redox transitions, which may underpin the redox-dependent modulation of partner binding by FNR. This integrated structural approach highlights the synergistic use of X-ray and neutron crystallography in studying redox-active proteins.

Differential scanning fluorimetry screening of the Library of Pharmacologically Active Compounds (LOPAC) identified four hits for the PRYSPRY domain of the human E3 ligase tripartite motif-containing protein 21 (TRIM21). Isothermal titration calorimetry subsequently confirmed suramin as a binder with micromolar affinity. To further investigate the binding mechanism, mouse TRIM21 was used as a structural surrogate due to its improved protein stability and high sequence similarity to the human counterpart. A crystal structure of the complex refined at 1.3 Å resolution revealed a unique binding mode, providing new avenues for targeting TRIM21 and for the development of proteolysis-targeting chimeras (PROTACs).

Polyamines are key signalling and substrate molecules that are made by all organisms. The polyamine known as spermidine is typically made by spermidine synthase, but in many bacterial species, including 70% of human gut microbes, carboxyspermidine decarboxylase (CASDC) performs the terminal step in the production of spermidine. An X-ray crystal structure of CASDC from the human gut microbe Clostridium leptum has been solved by molecular replacement at a resolution of 1.41 Å. CASDC is a homodimer, with each monomer composed of two domains: a β/α-barrel pyridoxal 5'-phosphate-binding domain that forms most of the active site and a β-barrel domain that extends the dimeric interface and contributes to the active site of the opposing monomer. We performed a structural comparison of CASDC enzymes for 15 common genera within the human gut flora. This analysis reveals structural differences occurring in the β6/β7 loop that acts as a `flap' covering the active site and in the α9/β12 loop that is connected to the α9 helix which is thought to select substrates by their chain length. This structural analysis extends our understanding of a key enzyme in spermidine biosynthesis in many bacterial species.

Helicobacter pylori is the primary causative agent of peptic ulcer disease, among other gastrointestinal ailments, and currently affects over half of the global population. Although some treatments exist, growing resistance to these drugs has prompted efforts to develop novel approaches to fighting this pathogen. To generate many of the nucleotides essential to biochemical processes, H. pylori relies exclusively on the de novo biosynthesis of these molecules. Recent drug-discovery efforts have targeted the first committed step of this pathway, catalysed by a class 2 dihydroorotate dehydrogenase (DHODH). However, these initiatives have been limited by the lack of a crystal structure. Here, we detail the crystal structure of H. pylori DHODH (HpDHODH) at 2.25 Å resolution (PDB entry 6b8s). We performed a large-scale bioinformatics search to find evolutionary homologs. Our results indicate that HpDHODH shows high conservation of both sequence and structure in its active site. We identified key polar interactions between the HpDHODH protein and its requisite flavin mononucleotide (FMN) cofactor, identifying amino-acid residues that are critical to its function. Most notably, we found that HpDHODH maintains several structural features that allow it to associate with the inner membrane and utilize ubiquinone to achieve catalytic turnover. We discovered a hydrophobic channel that runs from the putative membrane interface on the N-terminal microdomain to the core of the protein. We predict that this channel establishes a connection between the ubiquinone pool in the membrane and the FMN in the active site. These findings provide a structural explanation for the competitive inhibition of ubiquinone by pyrazole-based compounds that was determined biochemically in other studies. Understanding this mechanism may facilitate the development of new drugs targeting this enzyme and push the effort to find a resistance-free treatment for H. pylori.